Embryonic muscle development occurs through carefully orchestrated events that guide the progressive determination of each cell fate. At each stage of this process, regulatory networks operate through interactions of transcription factors (TFs) with specific cis regulatory modules (CRMs). This proposal specifically focuses on the homeodomain (HD) transcriptional regulatory networks governing progressive cell fate specificities of myoblast founder cell (FC) subtypes in the Drosophila embryo, where genes and molecular pathways are highly conserved up to humans. This proposal is comprised of three distinct, yet related parts. First, it will apply protein binding microarray (PBM) technology for high-throughput characterization of in vitro DNA binding specificities of mesodermally expressed TFs. The large motif dictionary of mesodermally expressed TFs not only will be essential for prediction of subset of cis regulatory codes promoting the formation of FCs, but also will serve as a valuable resource for the broader study of different cell types and tissue developments. Second, it will employ yeast two-hybrid (Y2H) assay to comprehensively identify HD cofactors. HD TFs have distinctive pattern of sequence specificity that can be altered by their protein binding activities. The resulting binding specificity data of these studies allow for improved prediction of genomic TF binding sites and TF's combinatorial co-regulation of target genes. Finally, a suite of computational tools (PhylCRM and Lever) will be used to predict combinations of HD TFs and their target CRMs driving gene expression during the specification of somatic muscle founder cell fate in the developing Drosophila mesoderm. To evaluate the potential PBM-derived and predicted cis regulatory codes, the effects of RNAi knockdown on the discovered enhancers will be examined. The specific aims will be: (1) To identify DNA binding specificities of 140 known and predicted TFs expressed in the Drosophila embryonic mesoderm. (2) To determine the DNA binding profiles of homo- and heterodimeric TFs. (3) To predict cell-type-specific CRMs and cis regulatory codes in Drosophila embryonic SM FCs and to experimentally validate the inferred cis regulatory codes by performing RNAi knockdown of the selected TFs. This study will be generally applicable to the identification of cis regulatory elements important in many different kinds of biological systems in any genome of interest. Furthermore, this information will permit a better understanding of regulatory DNA elements in vertebrate including humans and provide valuable insight into human muscle physiology and diseases. PUBLIC HEALTH RELEVANCE: This study will be generally applicable to the identification of cis regulatory elements important in many different kinds of biological systems in any genome of interest. Furthermore, this information will permit a better understanding of regulatory DNA elements in vertebrate including humans and provide valuable insight into human muscle physiology and diseases.